title: In vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with siRNAs targeting the spike glycoprotein and replicase polyprotein genes.
abstract: OBJECTIVE
  The specific effect of rna interference on the replication of porcine hemagglutinating encephalomyelitis virus (phe-cov) was explored.
  
  
  METHODS
  Four species of small interfering RNA (siRNA), targeting different regions of the PHE-CoV spike glycoprotein and replicase polyprotein genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHE-CoV, the cytopathic effect (CPE) was examined by phase-contrast microscope, and viral proliferation within cells was examined by indirect immunofluorescence microscopy, hemagglutination (HA) test, TCID(50) assay and real-time RT-PCR.
  
  
  RESULTS
  Examination of CPE demonstrated that the four siRNAs were capable of protecting cells against PHE-CoV invasion with very high specificity and efficiency. At 48 h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Transfection with siRNAs also suppressed the production of infectious virus by up to 18- to 32-fold as assessed by a HA test and 93- to 494-fold as assessed by TCID(50) assay. Furthermore, treatment with siRNAs caused a 53-91% reduction in the viral genome copy number as assessed by real-time RT-PCR.
  
  
  CONCLUSIONS
  These results suggested that the four species of siRNAs can efficiently inhibit PHE-CoV genome replication and infectious virus production.
authors.affiliation: College of Animal Science and Veterinary Medicine, Jilin University, Changchun, China.
authors.forename: Yungang
authors.initials: Y
authors.lastname: Lan
authors.forename: Huijun
authors.initials: H
authors.lastname: Lu
authors.forename: Kui
authors.initials: K
authors.lastname: Zhao
authors.forename: Wenqi
authors.initials: W
authors.lastname: He
authors.forename: Keyan
authors.initials: K
authors.lastname: Chen
authors.forename: Gaili
authors.initials: G
authors.lastname: Wang
authors.forename: Deguang
authors.initials: D
authors.lastname: Song
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D001688:Biological Products / Q000378:metabolism*; D002460:Cell Line; D017934:Coronavirus / Q000187:drug effects* / Q000254:growth & development*; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D006386:Hemagglutination Tests; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D008853:Microscopy; D020815:Polyproteins / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D060888:Real-Time Polymerase Chain Reaction; D064370:Spike Glycoprotein, Coronavirus; D013552:Swine; D014162:Transfection; D014759:Viral Envelope Proteins / Q000037:antagonists & inhibitors*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D001688:Biological Products; D008562:Membrane Glycoproteins; D020815:Polyproteins; D034741:RNA, Small Interfering; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D012324:RNA-Dependent RNA Polymerase